Mixed function oxidases from germinating castor bean endosperm

Homogenates from germinating castor bean endosperm were fractionated by sucrose density gradient centrifugation and examined for mixed function oxidase activity. Activity of cinnamic acid 4-hydroxylase and p-chloro-N-methylaniline N-demethylase was highest in the endoplasmic reticulum fraction. Activity of both enzymes is dependent on NADPH and on molecular oxygen; both activities are inhibited by carbon monoxide. When challenged with a number of potential inhibitors the enzymes responded in ways fairly typical of mixed function oxidases from other plants and animals. The N-demethylase appears to be specific for N-methylarylamines. In the absence of NADPH, cumene hydroperoxide is able to support N-demethylation. The mechanistic significance of this activity is discussed.     

Precursors of ricinine in the castor bean plant

Isotopic tracer experiments confirmed that glycerol and succinic acid are good precursors of the pyridine ring of ricinine in castor bean plants. Tritium from C-2 was lost from tritiated glycerol while tritium from C-1 was retained. Thus a derivative of dihydroxyacetone is likely to be intermediate. By simultaneous feeding of glycerol-1-(3)-[³H] and succinic acid-2(3)-[¹⁴C], it was hoped to find precursors of ricinine containing both labels, but none could be found. There was no evidence for the appearance of labeled quinolinic acid, which is presumed to be a precursor of ricinine.     

Endosperms and embryos of mature dry castor bean seeds contain active ribosomes

Mature dry endosperms and embryos of castor bean contain ribosomes which are capable of catalysing protein synthesis in vitro. This observation contradicts previous reports that ribosomes are absent from mature dry endosperms. On hydration of the mature seeds the polyribosome content of both embryos and endosperms increases. These polyribosomes are lost on subsequent dehydration but, again, ribosomes are conserved. REFERENCES     

Characterization of glyoxysomes from castor bean endosperm

Electron micrographs are presented which establish the identity of the components of the 3 major bands observed after sucrose density centrifugation of the crude particulate fraction from the endosperm of germinating castor bean seedlings. These are: mitochondria (density 1.19 g/cc), proplastids (density 1.23 g/cc) and glyoxysomes (density 1.25 g/cc). Further evidence is provided on the enzymatic composition of the glyoxysomes. Essentially all of the particulate malate synthetase, isocitrate lyase, catalase, and glycolic oxidase is present in these organelles. The distribution of glyoxysomal enzymes on sucrose density gradients is contrasted with that of the strictly mitochondrial enzymes fumarase, NADH oxidase, and succinoxidase. Malate dehydrogenase and citrate synthetase are present in both organelles. The functional role of glyoxysomes and their relationship to cytosomes from other tissues is discussed.     

我国优势油脂类生物柴油植物蓖麻的种质资源发掘和生物柴油利用

发展可再生生物质能源是解决人类能源危机和环境污染的重要途径。利用边际土地发展油脂类生物质能是生物质能的重要组成部分。蓖麻因为适应性强和油脂成份独特被誉为"理想的生物柴油植物"。蓖麻是我国优势油脂类能源植物,利用边际土地,发展蓖麻产业为我国生物柴油产业化提供原料,是我国现阶段生物柴油产业化发展的相对理想而又现实的选择,而且具有重要的发展前景和巨大的发掘潜力。立足我国现阶段生物柴油产业化的瓶颈问题,着重阐述了蓖麻种质资源发掘的现状、优良品种培育的途径和发展前景,以及利用蓖麻种子油生产商业化生物柴油的现状,以期推动我国利用边际土地发展蓖麻产业以及生物柴油商业化生产。     

Partial purification and characterization of CTP:cholinephosphate cytidylyltransferase from castor bean endosperm

CTP: cholinephosphate cytidylyltransferase (EC 2.7.7.15) has been purified approximately 600-fold from postgermination endosperm of castor bean. The enzyme was solubilized with n-octyl beta-D-glucopyranoside and then subjected to ion exchange and gel filtration chromatography. The Km's of the purified enzymatic activity were 0.37 and 1.1 mM for CTP and choline phosphate, respectively. Magnesium was required for activity. The purified cytidylyltransferase activity was inhibited by both phosphate and ATP. The extent of ATP inhibition was dependent on preincubation time, temperature, and Mg2+ and Ca2+ concentrations. The possible regulation of cytidylyltransferase in castor bean endosperm by protein phosphorylation is discussed.     

Partial purification and some regulatory properties of pyruvate kinase from germinating castor bean endosperm

The activity of pyruvate kinase in castor bean endosperm wasfound to increase rapidly from the 3rd to 4th day of germination.The crude enzyme is very labile, but, was stabilized by addingMg²⁺ with a high concentration of ethyleneglycol or glycerol.The enzyme thus stabilized was partially purified about 150-foldby fractionation with ammonium sulfate, and chromatography withDEAE-cellulose and Sephadex G-150. The enzyme showed normalMichaelis-Menten kinetics for both substrates; K_m for PEP andADP were 0.058 mM and 0.13 mM, respectively. Optimum pH wasnear 6.5. The effects of many kinds of amino acids, intermediatesof glycolysis, and the tricarboxylic acid cycle on the activityof the enzyme were tested. ATP was found to inhibit and -ketoglutarateto activate enzymatic activity. (Received December 2, 1975; )     

Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm

Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK_c, is localized in the cytosol, and the other, PK_p, is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K⁺ and Mg²⁺. Both isozymes are inhibited by citrate, pyruvate, and ATP. PK_c has a much broader pH profile than PK_p and is also more stable. Both have the same K_m (0.05 millimolar) for PEP, but PK_p has a 10-fold higher K_m (0.3 millimolar) for ADP than PK_c (0.03 millimolar). PK_c also has a higher affinity for alternate nucleotide substrates than PK_p. The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.     

Polymorphism of some glycosidases from barley

Barley grain extract displayed α-L-arabinosidase, β-fucosidase, β-galactosidase and β-glucosidase activities. Some of the glycosidases were separated from one another by Sephadex gel-filtration and CM-cellulose chromatography. The glycosidase types were more varied than reported by earlier workers. The multifunctional nature of some of the enzymes was demonstrated by inhibition studies, gel electrophoresis, pH and thermal stability studies.     

Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm

Metabolic pathways sequestered within the leucoplast of developing oilseeds ensure a balanced supply of substrates and cofactors for fatty acid biosynthesis. NADP-dependent malic enzyme (NADP-ME) may be important in supplying both carbon and NADPH for fatty acid biosynthesis in the developing endosperm of the oilseed Ricinus communis. NADP-ME was purified 5160-fold to a specific activity of 18.2 U/mg protein. NADP-ME is a homotetramer with a native mass of 254 kDa and a subunit size of approximately 63 kDa. Effectors of castor NADP-ME are typical of the NADP-malic enzymes, with the exception of acetyl-CoA and its derivatives, which were found to act as activators. This is consistent with a regulatory role for these molecules during fatty acid biosynthesis in vivo. NADP-ME was found to have maximal activity at stage 7 of endosperm development, coincident with maximal lipid accumulation.     

Purification of pyruvate kinase from germinating castor bean endosperm

Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.     

Lipid composition of organelles from germinating castor bean endosperm

Glyoxysome, endoplasmic reticulum, mitochondria, and proplastid fractions were isolated from endosperm of castor beans (Ricinus communis) germinated for 5 days at 30 C. Samples from sucrose density gradients were diluted with 0.15 m KCI and the membranes pelleted. Lipid extracts of these membranes were analyzed for phosphoglyceride, acyl lipid, and sterol content. The endoplasmic reticulum contains 1.24 mumol of phosphoglyceride per mg of protein; the mitochondria, 0.65 mumol/mg; and the glyoxysome membranes, 0.55 mumol/mg. Phosphatidyl choline and phosphatidyl ethanolamine are the most abundant lipids in all membranes studied, accounting for 70% or more of the lipid phosphorus and 50% or more of the fatty acid. Glyoxysome membranes and endoplasmic reticulum also contain phosphatidyl inositol (respectively, 9 and 17% of the lipid phosphorus) and free fatty acids (13% of the total fatty acid in each). Compared with other organelles, mitochondrial membranes have more phosphatidyl ethanolamine relative to phosphatidyl choline and are characterized by the presence of cardiolipin, in which 80% of the fatty acid is linoleate. The relative amounts of linoleate, palmitate, oleate, stearate, and linolenate in each of the phosphotoglycerides are constant regardless of the membrane source. Stimasgasterol and beta-sitosterol are present in the membranes (1-9 nmol each/mg protein).The data provide further evidence that glyoxysome membranes are derived from the endoplasmic reticulum but at the same time indicate some differentiation.     

Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated

Two catalase genes,cat1 andcat2, have been isolated from the castor bean genome. They were located in the same direction on a chromosome at a distance of 2.4 kb,cat1 being on the downstream side ofcat2. The two genes contained introns at the same positions except that one of the 7 introns incat1 is missing incat2 and the corresponding introns differed in size and sequence between the two genes. The translated regions of the two genes had the same number of nucleotides and exhibited 81.3% nucleotide sequence identity. In addition to introns, the nucleotide sequences of the 5-and 3-flanking regions are highly divergent between the two genes. In etiolated seedlings,cat1 mRNA was present abundantly in endosperms and cotyledons and only in a small amount in roots. Thecat1 mRNA could not be detected in hypocotyls. By contrast,cat2 mRNA is most abundant in hypocotyls and roots, while endosperms and cotyledons contained only low levels ofcat2 mRNA. Although neithercat1 norcat2 mRNA could be detected in dry seeds, both mRNAs showed temporal accumulation in the endosperm in response to germination. These results suggest that expression of two tightly linked catalase genes of castor bean,cat1 andcat2, are differentially regulated during development.     

Isolation of intact plastids from protoplasts from castor bean endosperm

Protoplasts were prepared from castor bean (Ricinus communis) endosperm by treatment with a mixture of the commercial enzymes Macerozyme R-10 and Cellulose “Onozuka” R-10. The protoplasts were gently ruptured by forcing the suspension through a hypodermic needle and the homogenate centrifuged on a linear sucrose gradient. From such a homogenate the mitochondria are recovered at their typical isopycnic density of 1.18 g/ml, but the glyoxysomes are retained, with other membranes, at a density of 1.13. The plastids reach their typical density of 1.22 on the gradient and are thus clearly separated from other organelles. Moreover, since essentially all of the ribulose bisphosphate carboxylase activity on the gradient is present in this fraction it can be concluded that the plastids are intact and have been recovered in high yield.     

Fungicidal effect of ultraviolet-C radiations in prevention of mycoflora of castor bean seeds

The effect of ultra violet (UV-C) radiation on germination and fungal flora of castor bean seeds was estimated with different doses stored up to 80?days interval. Results revealed that 20 and 40?min radiated castor bean seeds showed complete reduction in most of the fungi while germination of castor bean seeds were enhanced when UV-C radiations applied for 20?min. Increasing the radiation doses and storage period has been shown decrement in Aspergillus species colonisation.     

Diethylene glycol disulfide from castor bean cell suspension cultures

Diethylene glycol disulfide was isolated from castor bean cell suspension cultures. Incubation of suspension cultures with Na₂³⁵SO₄ resulted in the incorporation of radioactivity into the isolated diethylene glycol disulfide. Diethylene glycol disulfide was detected in cells (430 nmol/g cells) and in cell-free growth medium (41.5 nmol/ml).     

Nucleic acid (cDNA) and amino acid sequences of isocitrate lyase from castor bean

A cDNA library was constructed to mRNA enriched for isocitrate-lyase mRNA from castor-bean (Ricinus communis var. zanzibarensis) endosperms. Nine clones for isocitrate lyase (EC 4.1.3.1) were identified. The insert of 2.2 kb from clone ICL4 was sequenced and proved to contain the entire coding region, 1731 bp, for isocitrate lyase. The amino acid sequence of isocitrate lyase was deduced from the nucleic acid sequence. By analogy with muscle aldolase a lysine residue that possibly takes part in the binding of the substrate was identified. The 3 untranslated region contained three putative polyadenylation addition signals and two direct repeats.